CN104628848B - Monoclonal antibody MERS 27 and its encoding gene and application - Google Patents
Monoclonal antibody MERS 27 and its encoding gene and application Download PDFInfo
- Publication number
- CN104628848B CN104628848B CN201310565893.XA CN201310565893A CN104628848B CN 104628848 B CN104628848 B CN 104628848B CN 201310565893 A CN201310565893 A CN 201310565893A CN 104628848 B CN104628848 B CN 104628848B
- Authority
- CN
- China
- Prior art keywords
- mers
- cov
- sequence
- dna molecular
- chains
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 43
- 208000025370 Middle East respiratory syndrome Diseases 0.000 title abstract description 39
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 32
- 239000003814 drug Substances 0.000 claims abstract description 11
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 7
- 230000002265 prevention Effects 0.000 claims abstract description 6
- 230000009545 invasion Effects 0.000 claims abstract description 5
- 239000013612 plasmid Substances 0.000 claims description 47
- 241000127282 Middle East respiratory syndrome-related coronavirus Species 0.000 claims description 46
- 210000004027 cell Anatomy 0.000 claims description 33
- 239000000203 mixture Substances 0.000 claims description 15
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 10
- 201000010099 disease Diseases 0.000 claims description 9
- 239000002773 nucleotide Substances 0.000 claims description 8
- 125000003729 nucleotide group Chemical group 0.000 claims description 8
- 210000004962 mammalian cell Anatomy 0.000 claims description 7
- 208000015181 infectious disease Diseases 0.000 claims description 6
- 241000124008 Mammalia Species 0.000 claims description 4
- 125000000539 amino acid group Chemical group 0.000 abstract description 4
- 238000006467 substitution reaction Methods 0.000 abstract description 4
- 239000003795 chemical substances by application Substances 0.000 abstract description 2
- 230000000069 prophylactic effect Effects 0.000 abstract description 2
- 108020004414 DNA Proteins 0.000 description 31
- 230000000694 effects Effects 0.000 description 27
- 238000006386 neutralization reaction Methods 0.000 description 27
- 241001112090 Pseudovirus Species 0.000 description 24
- 239000007788 liquid Substances 0.000 description 24
- 241000700605 Viruses Species 0.000 description 19
- 239000000243 solution Substances 0.000 description 18
- 241000315672 SARS coronavirus Species 0.000 description 14
- 239000006228 supernatant Substances 0.000 description 12
- 238000011534 incubation Methods 0.000 description 10
- 230000031700 light absorption Effects 0.000 description 8
- 238000010790 dilution Methods 0.000 description 7
- 239000012895 dilution Substances 0.000 description 7
- 102000053602 DNA Human genes 0.000 description 6
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 6
- 239000005089 Luciferase Substances 0.000 description 6
- 239000001963 growth medium Substances 0.000 description 6
- 238000003780 insertion Methods 0.000 description 6
- 230000037431 insertion Effects 0.000 description 6
- 238000002360 preparation method Methods 0.000 description 6
- 101000600434 Homo sapiens Putative uncharacterized protein encoded by MIR7-3HG Proteins 0.000 description 5
- 102100037401 Putative uncharacterized protein encoded by MIR7-3HG Human genes 0.000 description 5
- 239000003153 chemical reaction reagent Substances 0.000 description 5
- 238000001514 detection method Methods 0.000 description 5
- 239000012634 fragment Substances 0.000 description 5
- 102100025012 Dipeptidyl peptidase 4 Human genes 0.000 description 4
- 238000008157 ELISA kit Methods 0.000 description 4
- 108010027044 HIV Core Protein p24 Proteins 0.000 description 4
- 101000908391 Homo sapiens Dipeptidyl peptidase 4 Proteins 0.000 description 4
- 108091028043 Nucleic acid sequence Proteins 0.000 description 4
- 239000000969 carrier Substances 0.000 description 4
- 238000013467 fragmentation Methods 0.000 description 4
- 238000006062 fragmentation reaction Methods 0.000 description 4
- 108091008146 restriction endonucleases Proteins 0.000 description 4
- 238000001890 transfection Methods 0.000 description 4
- 241000711573 Coronaviridae Species 0.000 description 3
- 150000001413 amino acids Chemical class 0.000 description 3
- 229940090124 dipeptidyl peptidase 4 (dpp-4) inhibitors for blood glucose lowering Drugs 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 230000003612 virological effect Effects 0.000 description 3
- 229920000936 Agarose Polymers 0.000 description 2
- 102100031673 Corneodesmosin Human genes 0.000 description 2
- 101710139375 Corneodesmosin Proteins 0.000 description 2
- 102000044437 S1 domains Human genes 0.000 description 2
- 108700036684 S1 domains Proteins 0.000 description 2
- 239000011324 bead Substances 0.000 description 2
- 238000009396 hybridization Methods 0.000 description 2
- 230000010355 oscillation Effects 0.000 description 2
- 108091033319 polynucleotide Proteins 0.000 description 2
- 102000040430 polynucleotide Human genes 0.000 description 2
- 239000002157 polynucleotide Substances 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 102000005962 receptors Human genes 0.000 description 2
- 108020003175 receptors Proteins 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 208000030507 AIDS Diseases 0.000 description 1
- 206010008342 Cervix carcinoma Diseases 0.000 description 1
- BWGNESOTFCXPMA-UHFFFAOYSA-N Dihydrogen disulfide Chemical compound SS BWGNESOTFCXPMA-UHFFFAOYSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 101001116436 Mus musculus Xaa-Pro dipeptidase Proteins 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 208000006105 Uterine Cervical Neoplasms Diseases 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 208000030303 breathing problems Diseases 0.000 description 1
- 201000010881 cervical cancer Diseases 0.000 description 1
- 238000002288 cocrystallisation Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 210000002919 epithelial cell Anatomy 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000002458 infectious effect Effects 0.000 description 1
- 201000007270 liver cancer Diseases 0.000 description 1
- 208000014018 liver neoplasm Diseases 0.000 description 1
- 238000003670 luciferase enzyme activity assay Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 238000000034 method Methods 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 239000002574 poison Substances 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000000197 pyrolysis Methods 0.000 description 1
- 238000012113 quantitative test Methods 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 229960005486 vaccine Drugs 0.000 description 1
- 239000002435 venom Substances 0.000 description 1
- 231100000611 venom Toxicity 0.000 description 1
- 210000001048 venom Anatomy 0.000 description 1
- 210000004885 white matter Anatomy 0.000 description 1
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention discloses a kind of monoclonal antibody MERS 27 and its encoding gene and application.Antibody provided by the invention, monoclonal antibody MERS 27 is named as, is made up of A chains and B chains；A chains are（a）Or（b）：（a）The protein being made up of the amino acid sequence shown in sequence 1；（b）Sequence 1 is passed through to substitution and/or missing and/or addition and the protein as derived from sequence 1 with identical function of one or several amino acid residues；B chains are（c）Or（d）：（c）The protein being made up of the amino acid sequence shown in sequence 3；（d）Sequence 3 is passed through to substitution and/or missing and/or addition and the protein as derived from sequence 3 with identical function of one or several amino acid residues.Monoclonal antibody provided by the invention can effectively suppress MERS CoV invasion cells, can be as MERS CoV prophylactic agent or medicine, and the prevention and treatment for MERS CoV are worth and be widely applied prospect with important theoretical direction.
Description
Technical field
The present invention relates to a kind of monoclonal antibody MERS-27 and its encoding gene and application.
Background technology
Novel coronavirus（MERS-CoV）It was found to infect the mankind, subsequent this disease in Middle East first in 2012
Disease caused by poison infection successively appears in the several countries and regions in Europe again.Infected patient more than half occurs seriously
Breathing problem, its clinical symptoms with 2003 outburst it is closely similar by SARS-CoV clinical symptoms.Due to this disease
Disease can be infected with people and given people, and cause global highest attention.Up to the present, also without specific medicine and vaccine pair
This disease is treated and prevented.
MERS-CoV utilizes the memebrane protein on its surface（S protein）Into permissive cell.S protein is by the S1 structures positioned at N-terminal
Domain and S2 domains and membrane spaning domain composition positioned at nearly film end, wherein virus is determined by S1 domains to cell susceptible
's.By using MERS-CoV S1 domains progress copurification experiment, Raj research group determines at the beginning of 2013
Dipeptideyl peptidase4 (DPP4, also referred to as CD26) are MERS-CoV acceptor.
The content of the invention
It is an object of the invention to provide a kind of monoclonal antibody MERS-27 and its encoding gene and application.
The invention provides a kind of antibody, is named as monoclonal antibody MERS-27, is made up of A chains and B chains；The A chains are
It is as follows（a）Or（b）：（a）The protein being made up of the amino acid sequence shown in sequence in sequence table 1；（b）By the amino of sequence 1
Acid sequence by one or several amino acid residues substitution and/or missing and/or addition and with identical function by sequence 1
Derivative protein；The B chains are as follows（c）Or（d）：（c）The egg being made up of the amino acid sequence shown in sequence in sequence table 3
White matter；（d）By the amino acid sequence of sequence 3 by the substitution and/or missing and/or addition of one or several amino acid residues and
With identical function as derived from sequence 3 protein.
The A chains and the B chains pass through disulfide formation monoclonal antibody MERS-27.
The present invention also protects a kind of DNA composition, by encoding the DNA molecular first of the A chains and encoding the DNA of the B chains
Molecule second forms.
The DNA molecular first can be as follows（1）Or（2）Or（3）DNA molecular：（1）Sequence 2 is from 5 ' ends in sequence table
DNA molecular shown in the nucleotides of 889-2301 positions；（2）Under strict conditions with（1）The DNA sequence dna hybridization of restriction and coding institute
State the DNA molecular of A chains；（3）With（1）The DNA sequence dna of restriction has the DNA molecular of more than 90% homology and the coding A chains.
Above-mentioned stringent condition can be in 6 × SSC, 0.5%SDS solution, hybridize under 65oC, then with 2 × SSC, 0.1%SDS and 1
× SSC, 0.1%SDS respectively washes film once.
The DNA molecular second is as follows（4）Or（5）Or（6）DNA molecular：（4）Sequence 4 is from 5 ' ends in sequence table
DNA molecular shown in the nucleotides of 889-1587 positions；（5）Under strict conditions with（4）Described in the DNA sequence dna hybridization of restriction and coding
The DNA molecular of B chains；（6）With（4）The DNA sequence dna of restriction has the DNA molecular of more than 90% homology and the coding B chains.On
It can be in 6 × SSC, 0.5%SDS solution to state stringent condition, hybridized under 65oC, then with 2 × SSC, 0.1%SDS and 1 ×
SSC, 0.1%SDS respectively wash film once.
The present invention also protects a kind of expression cassette composition, as described in expression cassette first and the expression of expressing the DNA molecular first
The expression cassette second composition of DNA molecular second.The expression cassette first specifically can be as shown in the sequence 2 of sequence table.Shown expression cassette second tool
Body can be as shown in the sequence 4 of sequence table.
The present invention also protects a kind of plasmid composition, is made up of recombinant plasmid first and recombinant plasmid second；The recombinant plasmid
First is the recombinant plasmid containing the DNA molecular first or the expression cassette first；The recombinant plasmid second is to contain the DNA molecular
The recombinant plasmid of second or the expression cassette second.Concretely the pMD18-T containing the expression cassette first is carried the recombinant plasmid first
Body.The recombinant plasmid second concretely contains the pMD18-T carriers of the expression cassette second.
The present invention also protects obtain the recombinant plasmid first and the recombinant plasmid second cotransfection mammalian cell
Recombinant cell.The mammalian cell concretely 293T cells.
Antibody (the IgG1 antibody that the recombinant cell obtains is cultivated in the present invention also protection）.
The present invention also protects a kind of medicine, and its active component is the monoclonal antibody MERS-27；The function of the medicine
To be as follows（Ⅰ）、（Ⅱ）、（Ⅲ）Or（Ⅳ）：（Ⅰ）Treatment and/or prevention MERS-CoV infection；（Ⅱ）Suppress MERS-CoV propagation；
（Ⅲ）Suppress MERS-CoV invasion mammals；（Ⅳ）Treat and/or prevent disease caused by MERS-CoV.It is described " to suppress
MERS-CoV breeds " concretely suppress propagation of the MERS-CoV in mammalian cell.The mammalian cell is specific
Can be Huh7 cells.
The present invention also protects applications of the monoclonal antibody MERS-27 in medicine is prepared；The function of the medicine is
It is as follows（Ⅰ）、（Ⅱ）、（Ⅲ）Or（Ⅳ）：（Ⅰ）Treatment and/or prevention MERS-CoV infection；（Ⅱ）Suppress MERS-CoV propagation；
（Ⅲ）Suppress MERS-CoV invasion mammals；（Ⅳ）Treat and/or prevent disease caused by MERS-CoV.It is described " to suppress
MERS-CoV breeds " concretely suppress propagation of the MERS-CoV in mammalian cell.The mammalian cell is specific
Can be Huh7 cells.
Monoclonal antibody provided by the invention can effectively suppress MERS-CoV invasion permissive cells, can be used as MERS-CoV
Prophylactic agent or medicine, the prevention and treatment for MERS-CoV with important theoretical direction value and widely should
Use prospect.
Brief description of the drawings
Fig. 1 is the SDS-PAGE of MERS-27 solution.
The step of Fig. 2 is embodiment 2 one to step 4 neutralization activity result；Ordinate is neutralization activity（%）, abscissa
For logarithm value of the protein concentration in dilution using e the bottom of as.
The neutralization activity result of the step of Fig. 3 is embodiment 2 five；Ordinate is neutralization activity（%）, abscissa is dilution
In logarithm value of the protein concentration using e the bottom of as.
Embodiment
Following embodiment facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method, it is conventional method unless otherwise specified.Test material used in following embodiments, unless otherwise specified,
It is to be commercially available from routine biochemistry reagent shop.Quantitative test in following examples, is respectively provided with and weighs three times
Multiple experiment, results averaged.
293T cells（People's renal epithelial cell system）：ATCC,CRL-11268TM.Huh7 cells（Liver cancer cell lines）：
JCRB, JCR B0403.TZM-BL cells (cervical cancer tumer line)：NIH AIDS Research and Reference
Reagent Program(Cat.No.8129).PMD18-T carriers：Takara.PcDNA3.1 (+) carrier：Invitrogen is public
Department.Skeleton plasmid pNL4-3R-E-luciferase：Bibliography:He J,Choe S,Walker R,Di Marzio P,
Morgan DO,Landau NR.J Virol69:6705–6711,1995.。
The discovery of embodiment 1, monoclonal antibody
Find that MERS-CoV there may be a potential receptor binding domains (Receptor by sequence and model analysis
Binding Domain, RBD), by RBD and soluble DPP4 albumen cocrystallization, parse the structure of albumen, find to have in RBD with
The key amino acid site that DPP4 is combined.Animal immune is carried out using the RBD of purifying, it is found that it is anti-there is stronger induction to neutralize for it
Ability caused by body.The nonimmune scFvs libraries of the mankind of yeast surface are illustrated in using RBD screenings（ScFvs English full name is
“single-chain variable fragments”）, obtained some monoclonal antibodies with neutralization activity.Will wherein
One monoclonal antibody is named as monoclonal antibody MERS-27（Belong to IgG1 antibody）.
The preparation of embodiment 2, monoclonal antibody MERS-27
1st, the double chain DNA fragment first shown in the sequence 2 of composition sequence table, then DNA fragmentation first insertion pMD18-T is carried
Body, obtain recombinant plasmid first.
It is CMV promoter from 5 ' end 1-888 positions nucleotides in the sequence 2 of sequence table, 889-2301 positions nucleotides
For the encoding gene of monoclonal antibody MERS-27 heavy chain, 2302-2429 is ployA.It is double shown in the sequence 2 of sequence table
Protein shown in the sequence 1 of ssdna molecule polynucleotide.
2nd, the double chain DNA fragment second shown in the sequence 4 of composition sequence table, then DNA fragmentation second insertion pMD18-T is carried
Body, obtain recombinant plasmid second.
It is CMV promoter from 5 ' end 1-888 positions nucleotides in the sequence 4 of sequence table, 889-1587 positions nucleotides
For the encoding gene of monoclonal antibody MERS-27 light chain, 1588-1715 is ployA.It is double shown in the sequence 4 of sequence table
Protein shown in the sequence 3 of ssdna molecule polynucleotide.
3rd, by recombinant plasmid first and recombinant plasmid second cotransfection 293T cells（Transfect dosage：Every 1 × 105Individual cell transfecting 2
Microgram recombinant plasmid first and 2 microgram recombinant plasmid second, the culture medium used is the DMEM culture medium containing 10%FBS）, 37 DEG C of standings
It is incubated 8 hours, culture medium is then replaced by the DMEM culture mediums containing 2%FBS and 37 DEG C of stationary incubations 72 hours（Practical application
In, 48-72 hours）, then receive cells and supernatant, 4 DEG C, 4000rpm centrifuge 1 hour, collect supernatant.
4th, the supernatant for taking step 3 to obtain, protein A beads are added（PierceTMProtein A Agarose；
Thermo companies）, 4 DEG C of oscillation incubations 12 hours, centrifuging and taking supernatant, the as solution containing monoclonal antibody MERS-27（Letter
Claim MERS-27 solution）, 4 DEG C of preservations.
The SDS-PAGE of MERS-27 solution is shown in Fig. 1.By Fig. 1 it is observed that having no it in MERS-27 solution
Its foreign protein.Two bands are separately recovered and are sequenced, before the preceding 10 amino acids residue of a band is the sequence 1 of sequence table
10, the preceding 10 amino acids residue of alternative in vitro test is first 10 of the sequence 3 of sequence table.
The application of embodiment 3, monoclonal antibody MERS-27
First, neutralization activities of the monoclonal antibody MERS-27 to MERS-CoV pseudovirus is detected
Express the plasmid of MERS-CoV total length memebrane proteins（It is named as MERS-CoV memebrane protein plasmids）With skeleton plasmid pNL4-
3R-E-luciferase cotransfection 293T cells, it can obtain there is infectivity but without the MERS- of replication capacity after incubation
CoV pseudotype virus, it is infectious similar with live virus.
By the double chain DNA molecule shown in the sequence 5 of sequence table（The encoding gene of MERS-CoV total length memebrane proteins）Insertion
Between HindIII the and XhoI restriction enzyme sites of pcDNA3.1 (+) carrier, MERS-CoV memebrane protein plasmids are obtained.
1st, the preparation of MERS-CoV pseudovirus
By MERS-CoV memebrane proteins plasmid and skeleton plasmid pNL4-3R-E-luciferase cotransfection 293T cells, 37 DEG C
Stationary incubation, transfection collect cells and supernatant, the as virus liquid containing MERS-CoV pseudovirus after 48 hours（Referred to as
MERS-CoV virus liquids）.The ELISA kit quantitatively detected using p24（HIV P24 antigen immue quantitative detection reagent boxes, KEY-
BIO,96T）Detect the virus titer of MERS-CoV virus liquids, the OD of MERS-CoV virus liquids450nm（Light absorption value is 1
（1021TCID50/ml）, light absorption value is bigger, and explanation viral level is higher.
2nd, neutralization activities of the monoclonal antibody MERS-27 to MERS-CoV pseudovirus is detected
（1）The MERS-27 solution doubling dilutions that embodiment 2 is prepared using the DMEM culture mediums containing 10%FBS, according to
It is secondary obtain protein concentration for 50.000000 μ g/ml, 16.666670 μ g/ml, 5.555555 μ g/ml, 1.851852 μ g/ml,
0.6172839μg/ml、0.2057613μg/ml、0.06858711μg/ml、0.02286237μg/ml、0.00762079μg/
ml、0.002540263μg/ml、0.000846754μg/ml、0.000282251μg/ml、0.0000940838μg/ml、
0.0000313613 μ g/ml, 0.0000104538 μ g/ml and 0.00000348459 μ g/ml dilution.
（2）By 100 microlitres of steps（1）Obtained dilution mixes with the MERS-CoV virus liquids that 50 microlitres of steps 1 obtain,
37 DEG C of stationary incubations 1 hour；The blank pair that 100 microlitres of dilutions are replaced with 100 microlitres of DMEM culture mediums containing 10%FBS is set
According to.
（3）Complete step（2）Afterwards, the cell liquid of 50 microlitres of Huh7 cells is added（Containing about 2 × 104Individual Huh7 cells）, 37
DEG C stationary incubation 48 hours（In practical application, 48-72 hours）.
（4）Complete step（3）Afterwards, 100 μ l PBSs and 50 μ l cell pyrolysis liquids are added（Bright-
GloTMLuciferase Assay System, Promega, E2650）, 2min is stood, then detects fluorescence with Chemiluminescence Apparatus
Plain enzymatic activity.
Every kind of processing sets 5 multiple holes, results averaged.
Neutralization activity=（The fluorescence intensity of the experimental group of the fluorescence intensity of blank control group-addition dilution）/ blank control
Fluorescence intensity × 100% of group.
The result of neutralization activity is shown in Fig. 2.
2nd, neutralization activities of the monoclonal antibody MERS-27 to SARS-CoV pseudovirus is detected
By the double chain DNA molecule shown in the sequence 6 of sequence table（The encoding gene of SARS-CoV total length memebrane proteins）Insertion
Between HindIII the and XhoI restriction enzyme sites of pcDNA3.1 (+) carrier, SARS-CoV memebrane protein plasmids are obtained.
1st, the preparation of SARS-CoV pseudovirus
By SARS-CoV memebrane proteins plasmid and skeleton plasmid pNL4-3R-E-luciferase cotransfection 293T cells, 37 DEG C
Stationary incubation, transfection collect cells and supernatant, the as virus liquid containing SARS-CoV pseudovirus after 48 hours（Referred to as
SARS-CoV virus liquids）.The ELISA kit quantitatively detected using p24（HIV P24 antigen immue quantitative detection reagent boxes, KEY-
BIO,96T）Detect the virus titer of SARS-CoV virus liquids, the OD of SARS-CoV virus liquids450nm（Light absorption value is 1
（1021TCID50/ml）, light absorption value is bigger, and explanation viral level is higher.
2nd, neutralization activities of the monoclonal antibody MERS-27 to SARS-CoV pseudovirus is detected
MERS-CoV virus liquids are replaced with SARS-CoV virus liquids, the 2 of other Complete Synchronizations rapid one.
The result of neutralization activity is shown in Fig. 2.
3rd, neutralization activities of the monoclonal antibody MERS-27 to VSVG pseudovirus is detected
By the double chain DNA molecule shown in the sequence 7 of sequence table（The encoding gene of VSVG total length memebrane proteins）Insertion
Between HindIII the and XhoI restriction enzyme sites of pcDNA3.1 (+) carrier, VSVG memebrane protein plasmids are obtained.
1st, the preparation of VSVG pseudovirus
By VSVG memebrane proteins plasmid and skeleton plasmid pNL4-3R-E-luciferase cotransfection 293T cells, 37 DEG C of standings
It is incubated, transfection collects cells and supernatant, the as virus liquid containing VSVG pseudovirus after 48 hours（Abbreviation VSVG virus liquids）.
The ELISA kit quantitatively detected using p24（HIV P24 antigen immue quantitative detection reagent boxes, KEY-BIO, 96T）Detect VSVG diseases
The virus titer of venom, the OD of VSVG virus liquids450nm（Light absorption value is 1（1021TCID50/ml）, the bigger explanation virus of light absorption value
Content is higher.
2nd, neutralization activities of the monoclonal antibody MERS-27 to VSVG pseudovirus is detected
MERS-CoV virus liquids are replaced with VSVG virus liquids, the 2 of other Complete Synchronizations rapid one.
The result of neutralization activity is shown in Fig. 2.
4th, neutralization activities of the monoclonal antibody MERS-27 to CNE11 pseudovirus is detected
By the double chain DNA molecule shown in the sequence 8 of sequence table（The encoding gene of CNE11 total length memebrane proteins）Insertion
Between HindIII the and XhoI restriction enzyme sites of pcDNA3.1 (+) carrier, CNE11 memebrane protein plasmids are obtained.
1st, the preparation of CNE11 pseudovirus
By CNE11 memebrane proteins plasmid and skeleton plasmid pNL4-3R-E-luciferase cotransfection 293T cells, 37 DEG C quiet
Incubation is put, transfection collects cells and supernatant, the as virus liquid containing CNE11 pseudovirus after 48 hours（Abbreviation CNE11 viruses
Liquid）.The ELISA kit quantitatively detected using p24（HIV P24 antigen immue quantitative detection reagent boxes, KEY-BIO, 96T）Detection
The virus titer of CNE11 virus liquids, the OD of CNE11 virus liquids450nm（Light absorption value is 1（1021TCID50/ml）, light absorption value is bigger
Illustrate that viral level is higher.
2nd, neutralization activities of the monoclonal antibody MERS-27 to CNE11 pseudovirus is detected
MERS-CoV virus liquids are replaced with CNE11 virus liquids, the 2 of other Complete Synchronizations rapid one.
The result of neutralization activity is shown in Fig. 2.
5th, detect monoclonal antibody VRC01 to MERS-CoV pseudovirus, SARS-CoV pseudovirus, VSVG pseudovirus and
The neutralization activity of CNE11 pseudovirus
1st, monoclonal antibody VRC01 preparation
（1）Double chain DNA fragment shown in the sequence 9 of composition sequence table, DNA fragmentation is then inserted into pMD18-T carriers, obtained
To recombinant plasmid third.
（2）Double chain DNA fragment shown in the sequence 10 of composition sequence table, DNA fragmentation is then inserted into pMD18-T carriers,
Obtain recombinant plasmid fourth.
（3）By recombinant plasmid third and recombinant plasmid fourth cotransfection 293T cells（Transfect dosage：Every 1 × 105Individual cell transfecting
2 microgram recombinant plasmids third and 2 microgram recombinant plasmid fourths, the culture medium used is the DMEM culture medium containing 10%FBS）, 37 DEG C of standings
It is incubated 8 hours, culture medium is then replaced by the DMEM culture mediums containing 2%FBS and 37 DEG C of stationary incubations 72 hours（Practical application
In, 48-72 hours）, then receive cells and supernatant, 4 DEG C, 4000rpm centrifuge 1 hour, collect supernatant.
（4）Take step（3）Obtained supernatant, add protein A beads（PierceTMProtein A
Agarose；Thermo companies）, 4 DEG C of oscillation incubations 12 hours, centrifuging and taking supernatant, as containing monoclonal antibody VRC01's
Solution（Abbreviation VRC01 solution）, 4 DEG C of preservations.
2nd, neutralization activities of the monoclonal antibody VRC01 to MERS-CoV pseudovirus is detected
MERS-27 solution, other same step 1 are replaced with VRC01 solution.
The result of neutralization activity is shown in Fig. 3.
3rd, neutralization activities of the monoclonal antibody VRC01 to SARS-CoV pseudovirus is detected
MERS-27 solution, other same step 2 are replaced with VRC01 solution.
The result of neutralization activity is shown in Fig. 3.
4th, neutralization activities of the monoclonal antibody VRC01 to VSVG pseudovirus is detected
MERS-27 solution, other same step 3 are replaced with VRC01 solution.
The result of neutralization activity is shown in Fig. 3.
5th, neutralization activities of the monoclonal antibody VRC01 to CNE11 pseudovirus is detected
MERS-27 solution, other same step 4 are replaced with VRC01 solution.
The result of neutralization activity is shown in Fig. 3.
Result of this example indicate that monoclonal antibody MERS-27 provided by the invention can be with specific suppression MERS-
CoV propagation.
Claims (9)
1. a kind of antibody, it is made up of A chains and B chains；
The A chains are the protein being made up of the amino acid sequence shown in sequence in sequence table 1；
The B chains are the protein being made up of the amino acid sequence shown in sequence in sequence table 3.
2.DNA compositions, by the B in the DNA molecular first and coding claim 1 of the A chains in coding claim 1
The DNA molecular second composition of chain.
3. DNA composition as claimed in claim 2, it is characterised in that：The DNA molecular first is sequence 2 from 5 ' in sequence table
DNA molecular shown in the nucleotides of end 889-2301 positions；The DNA molecular second be in sequence table sequence 4 from 5 ' ends the
DNA molecular shown in the nucleotides of 889-1587 positions.
4. a kind of expression cassette composition, weighed by the expression cassette first and expression of the DNA molecular first in expression Claims 2 or 3
Profit requires the expression cassette second composition of the DNA molecular second in 2 or 3.
5. a kind of plasmid composition, it is made up of recombinant plasmid first and recombinant plasmid second；The recombinant plasmid first is to be wanted containing having the right
Ask the DNA molecular first in 2 or 3 or the recombinant plasmid containing the expression cassette first in claim 4；The recombinant plasmid
Second is to contain the DNA molecular second in Claims 2 or 3 or the restructuring matter containing the expression cassette second in claim 4
Grain.
6. the weight that the recombinant plasmid first in claim 5 and the recombinant plasmid second cotransfection mammalian cell are obtained
Group cell.
7. the antibody that recombinant cell described in culture claim 6 obtains.
8. a kind of medicine, its active component is the antibody described in claim 1；The function of the medicine is as follows（Ⅰ）、（Ⅱ）、
（Ⅲ）Or（Ⅳ）：（Ⅰ）Treatment and/or prevention MERS-CoV infection；（Ⅱ）Suppress MERS-CoV propagation；（Ⅲ）Suppress MERS-CoV
Invade mammal；（Ⅳ）Treat and/or prevent disease caused by MERS-CoV.
9. application of the antibody described in claim 1 in medicine is prepared；The function of the medicine is as follows（Ⅰ）、（Ⅱ）、（Ⅲ）
Or（Ⅳ）：（Ⅰ）Treatment and/or prevention MERS-CoV infection；（Ⅱ）Suppress MERS-CoV propagation；（Ⅲ）Suppress MERS-CoV invasions
Mammal；（Ⅳ）Treat and/or prevent disease caused by MERS-CoV.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310565893.XA CN104628848B (en) | 2013-11-14 | 2013-11-14 | Monoclonal antibody MERS 27 and its encoding gene and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310565893.XA CN104628848B (en) | 2013-11-14 | 2013-11-14 | Monoclonal antibody MERS 27 and its encoding gene and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104628848A CN104628848A (en) | 2015-05-20 |
| CN104628848B true CN104628848B (en) | 2018-01-23 |
Family
ID=53208130
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310565893.XA Active CN104628848B (en) | 2013-11-14 | 2013-11-14 | Monoclonal antibody MERS 27 and its encoding gene and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104628848B (en) |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106478814A (en) * | 2015-08-25 | 2017-03-08 | 复旦大学 | One kind goes the full human monoclonal antibody of fucosylation and its application |
| CN105859882B (en) * | 2016-04-13 | 2021-07-20 | 中国医学科学院病原生物学研究所 | Humanized neutralizing antibody A1 for resisting MERS virus, and preparation method and application thereof |
| CN107298712B (en) * | 2016-04-14 | 2020-04-07 | 中国疾病预防控制中心病毒病预防控制所 | Fully humanized neutralizing antibody against middle east respiratory syndrome coronavirus |
| CN106380517B (en) * | 2016-10-28 | 2019-07-16 | 中国人民解放军军事医学科学院微生物流行病研究所 | A kind of pair of Middle East respiration syndrome coronavirus has small molecular antibody and its application of neutralization activity |
| CN109666070B (en) * | 2017-10-13 | 2021-02-19 | 清华大学 | Monoclonal antibody MERS-4V2 and coding gene and application thereof |
| WO2021203397A1 (en) * | 2020-04-10 | 2021-10-14 | Tsb Therapeutics (Beijing) Co. Ltd. | Anti-sars-cov-2 antibodies and uses thereof |
| CN116240175B (en) * | 2023-02-28 | 2024-02-23 | 武汉科技大学 | Preparation method of chimeric anti-HIV broad-spectrum neutralizing antibody exosome and application thereof in anti-HIV infection |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1852921A (en) * | 2003-07-21 | 2006-10-25 | 美国政府卫生及公众服务部国家卫生协会 | Soluble fragments of the SARS-cov spike glycoprotein |
| CN1911963A (en) * | 2005-08-10 | 2007-02-14 | 中国科学院上海生命科学研究院 | SARS neutralization antibody and application |
| CN1914226A (en) * | 2003-11-25 | 2007-02-14 | 达纳-法伯癌症研究院有限公司 | Antibodies against SARS-COV and methods of use thereof |
| CN101098710A (en) * | 2004-06-02 | 2008-01-02 | 纽约血液中心 | Sars vaccines and methods to produce highly potent antibodies |
| CN101522208A (en) * | 2005-02-08 | 2009-09-02 | 纽约血库公司 | Neutralizing monoclonal antibodies against severe acute respiratory syndrome-associated coronavirus |
| CN102015767A (en) * | 2008-01-17 | 2011-04-13 | 胡马斯有限公司 | Cross-neutralizing human monoclonal antibodies to SARS-CoV and methods of use thereof |
| CN102690336A (en) * | 2012-06-11 | 2012-09-26 | 中国科学院武汉病毒研究所 | Bat SARS-like coronavirus spike protein immunity determining area and preparation method and application thereof |
-
2013
- 2013-11-14 CN CN201310565893.XA patent/CN104628848B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1852921A (en) * | 2003-07-21 | 2006-10-25 | 美国政府卫生及公众服务部国家卫生协会 | Soluble fragments of the SARS-cov spike glycoprotein |
| CN1914226A (en) * | 2003-11-25 | 2007-02-14 | 达纳-法伯癌症研究院有限公司 | Antibodies against SARS-COV and methods of use thereof |
| CN101098710A (en) * | 2004-06-02 | 2008-01-02 | 纽约血液中心 | Sars vaccines and methods to produce highly potent antibodies |
| CN101522208A (en) * | 2005-02-08 | 2009-09-02 | 纽约血库公司 | Neutralizing monoclonal antibodies against severe acute respiratory syndrome-associated coronavirus |
| CN1911963A (en) * | 2005-08-10 | 2007-02-14 | 中国科学院上海生命科学研究院 | SARS neutralization antibody and application |
| CN102015767A (en) * | 2008-01-17 | 2011-04-13 | 胡马斯有限公司 | Cross-neutralizing human monoclonal antibodies to SARS-CoV and methods of use thereof |
| CN102690336A (en) * | 2012-06-11 | 2012-09-26 | 中国科学院武汉病毒研究所 | Bat SARS-like coronavirus spike protein immunity determining area and preparation method and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104628848A (en) | 2015-05-20 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104628848B (en) | Monoclonal antibody MERS 27 and its encoding gene and application | |
| Alvarez et al. | HIV-1 Vpu antagonism of tetherin inhibits antibody-dependent cellular cytotoxic responses by natural killer cells | |
| Wang et al. | 7SL RNA mediates virion packaging of the antiviral cytidine deaminase APOBEC3G | |
| Usami et al. | SERINC3 and SERINC5 restrict HIV-1 infectivity and are counteracted by Nef | |
| Ellwanger et al. | Exosomes in HIV infection: A review and critical look | |
| CN104628849B (en) | Monoclonal antibody MERS 4 and its encoding gene and application | |
| Bonsignori et al. | Two distinct broadly neutralizing antibody specificities of different clonal lineages in a single HIV-1-infected donor: implications for vaccine design | |
| Poignard et al. | Heterogeneity of envelope molecules expressed on primary human immunodeficiency virus type 1 particles as probed by the binding of neutralizing and nonneutralizing antibodies | |
| Peters et al. | Non-macrophage-tropic human immunodeficiency virus type 1 R5 envelopes predominate in blood, lymph nodes, and semen: implications for transmission and pathogenesis | |
| Popgeorgiev et al. | Marseillevirus-like virus recovered from blood donated by asymptomatic humans | |
| Gray et al. | Isolation of a monoclonal antibody that targets the alpha-2 helix of gp120 and represents the initial autologous neutralizing-antibody response in an HIV-1 subtype C-infected individual | |
| Durham et al. | Neutralization resistance of virological synapse-mediated HIV-1 Infection is regulated by the gp41 cytoplasmic tail | |
| Peters et al. | Variation in HIV-1 R5 macrophage-tropism correlates with sensitivity to reagents that block envelope: CD4 interactions but not with sensitivity to other entry inhibitors | |
| Gessain | Human retrovirus HTLV-1: descriptive and molecular epidemiology, origin, evolution, diagnosis and associated diseases | |
| Krumm et al. | Mechanisms of escape from the PGT128 family of anti-HIV broadly neutralizing antibodies | |
| Veenhuis et al. | Long-term remission despite clonal expansion of replication-competent HIV-1 isolates | |
| Ritchie et al. | Analysis of HIV-1 Gag protein interactions via biotin ligase tagging | |
| Montefiori et al. | Antibody-dependent enhancement of simian immunodeficiency virus (SIV) infection in vitro by plasma from SIV-infected rhesus macaques | |
| Welch et al. | Vehicles of intercellular communication: exosomes and HIV-1 | |
| Olinger et al. | CD4-Negative cells bind human immunodeficiency virus type 1 and efficiently transfer virus to T cells | |
| Gramberg et al. | Evidence for an activation domain at the amino terminus of simian immunodeficiency virus Vpx | |
| Cachat et al. | Alpha interferon restricts human T-lymphotropic virus type 1 and 2 de novo infection through PKR activation | |
| Gach et al. | Human immunodeficiency virus type-1 (HIV-1) evades antibody-dependent phagocytosis | |
| WO2001059457A3 (en) | Assay for detection of viral fusion inhibitors | |
| Ollerton et al. | HIV-1-specific chimeric antigen receptor T cells fail to recognize and eliminate the follicular dendritic cell HIV reservoir in vitro |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |